Despite several reports of HIV-1-infected alveolar macrophages (AM) in the lungs of HIV-1-infected individuals, the roles played by these cells in the maintenance or persistence of infection remain unresolved. Recent studies in Malawi, conducted on AM from HIV-1-infected individuals that are effectively virally- suppressed by long-term ART, reproducibly detected the presence of HIV-1 mRNA by fluorescent in situ hybridization (FISH). In these studies, we also detected HIV-1 transcripts through single cell sequencing protocols, and have isolated infectious HIV-1 virus from cells recovered by bronchoalveolar lavage from ART- nave, HIV-1-infected volunteers. The hypothesis we propose to test is that the presence of HIV-1 transcripts in the alveolar macrophages of HIV- infected individuals is indicative of a productive viral infection that has significance for persistence of the virus during ART. R21 Phase: Aim 1. Are HIV-1-infected AM productively infected? We propose co-culture approaches to detect and capture infectious HIV-1 from the AM and peripheral blood of HIV-1-infected volunteers in Malawi. Using permissive, HIV-1-susceptible reporter cells we have already shown that we can acquire infectious virus from ART-nave individuals in a pilot study on 12 volunteers. We propose expanding this analysis to a larger cohort, including ART-treated, virally-suppressed individuals. R33 Phase: Aim 2. Transcriptional Profiling of Viral and Host Transcripts in HIV-1-Infected Cells. Using methods already established in Malawi, we will generate transcriptional profiles of HIV-infected and uninfected AMs by single-cell Seq-Well and Flow-FISH RNASeq methods to obtain datasets reflecting both single-cell resolution and depth of coverage. We will use these datasets to probe the impact of HIV-1 in cell longevity and to study the cellular tropism of the envs from AM-derived HIV-1 virus. R33 Phase: Aim 3. Perturbation of HIV-1-infected AM Function with Synthetic mRNA and SiRNA. We will use gain-of-function (synthetic mRNA) and loss-of-function (siRNA) approaches to manipulate the phenotype and behavior of HIV-1-infected HMDMs and AMs. The goal is to identify pathways that will drive programmed cell death specifically in those AMs that are HIV-1-infected as a route for selective eradication of this potential viral reservoir. The proposal is based on the contention that AM in virally-suppressed individuals will prove to be productively- infected. The verification of this contention is the major milestone of the R21 period of this phased award.